The NF-AT transcription factors are key effectors in the calcium signaling pathways underlying T cell activation, muscle development, and synaptic plasticity. It is generally thought that the four NF-ATs are cytoplasmic in resting cells but triggered to enter the nucleus and transactivate genes by calcineurin, a calciumactivated phosphatase. Recent data, however, are overturning some of the assumptions of the NF-AT model and offering new insights to the complex regulatory mechanisms governing this arm of calcium signaling. For instance, mouse gene knockouts of the four NF-AT isotypes have rendered the unexpected conclusion that NFAT1 and NF-AT4 are in fact negative regulators of T cell activation, while only NF-AT2 appears to have the predicted positive activity towards cytokine genes. These data are especially interesting because T cells, myocytes, and neurons appear to simultaneously express multiple NF-AT isotypes, indicating that activation must depend on the differential regulation of these factors. We have now begun to address this problem by evaluating the dynamics of all four NE-AT isotypes as a function of calcium signaling and cellular environment. Significantly, these data reveal a remarkable degree of NE-AT isotype-dependent dynamics and suggest important contributions of cell type and calcium-independent pathways to the ultimate NF-AT response. We hypothesize, therefore, that these NF-AT family members must be differentially subject to regulatory pathways that override or extend those imparted by calcium signaling, and that understanding these alternative mechanisms is essential for predicting cellular functions dependent on NF-ATs. This is a proposal to uncover the mechanisms of differential NE-AT signaling in the cell. We will (1), determine the biochemical signaling pathways underlying differential NE-AT regulation and their extent; (2) elucidate the calcium-independent pathways that operate through a highly conserved activation mechanism conserved in all four NE-ATs; (3), identify small molecules through high-throughput optical screens of NE-AT dynamics which differentially modulate the NE-AT isotypes; and (4), use these small molecules to functionally dissect the individual contributions of NE-AT isotypes to complex processes such as T cell activation. We anticipate that this novel evaluation of NE-AT activity will directly contribute to more specific therapies for autoimmune disease, organ transplantation, and cardiomyopathies.